Full genome sequence of a peste des petits ruminants virus (PPRV) from Ghana.

The full genome of a peste des petits ruminants virus (PPRV) isolated from a sheep lung sample collected in Ghana, Western Africa, in 2010, has been sequenced. Phylogenetic analysis demonstrated that the virus clustered within the lineage II clade while comparison of its full genome with those of other PPRV strains revealed the highest identity (96.6 %) at a nucleotide level with the PPRV strain Nigeria/76/1. This is the first full genome sequence generated for a PPRV lineage II isolated since 1976.

First experiences with a combined usage of veno-arterial and veno-venous ECMO in therapy-refractory cardiogenic shock patients with cerebral hypoxemia.

The use of extracorporeal membrane oxygenation (ECMO) is becoming a popular tool in the treatment of cardiogenic shock. We present two case reports where classical veno-arterial peripherally cannulated ECMO therapy proved insufficient with profuse cerebral hypoxemia. After augmenting the setting into veno-veno-arterial ECMO, we achieved a remarkable improvement of all oxygenation parameters. The simultaneous use of veno-venous and veno-arterial ECMO might display as a novel strategy to counteract the coronary and cerebral hypoxemia in veno-arterial ECMO therapy in patients with therapy-refractory cardiogenic shock or in combined cardiopulmonary failure. In this manuscript, the veno-veno-arterial ECMO setup is described in full detail and different venous cannulas are discussed.

Intraoperative sRAGE kinetics. A new age-related outcome predictor of cardiac surgery.

BACKGROUND: Glycated proteins (advanced glycation endproducts, AGE) in tissue are associated with degenerative diseases. This study evaluated the role of sRAGE (soluble receptor for advanced glycation endproducts), a decoy receptor of AGEs in blood, for the outcome of patients after coronary artery bypass grafting (CABG). METHODS: A total of 90 patients undergoing CABG were analysed in two centres. Perioperative blood samples were collected before surgery up to 1 week postoperatively. sRAGE was measured by ELISA. Patients were subdivided regarding age (< 64 versus > 70 years, 14 % versus 35 % female), euroSCORE (< 3 versus > 4, 14 % versus 29 % female) and sRAGE changes between sternotomy and end of the operation (< 30 % versus > 45 %, 33 % versus 33 % female) and subsequently analysed with respect of postoperative outcome parameters. RESULTS: Preoperative sRAGE values did not correlate with the outcome of the patients. sRAGE levels increase within 10 min from 1,539 ± 96 to 5,311 ± 187 pg/ml after sternotomy, then returning to baseline levels within 2 days after surgery. Comparing the analysed possible risk factors age, euroSCORE and sRAGE changes, no difference was observed regarding 30-day mortality. Age and the euroSCORE are superior with respect of tachyarrythmia, whereas sRAGE kinetics seems to be superior with respect of prolonged postoperative respiration time/stay in the intensive care unit or catecholamine support. CONCLUSION: A prolonged, increased intraoperative sRAGE level is a new outcome predictor for patients undergoing CABG surgery, mutually complementary to the euroSCORE.

Immediate effects of individualized heparin and protamine management on hemostatic activation and platelet function in adult patients undergoing cardiac surgery with tranexamic acid antifibrinolytic therapy.

OBJECTIVE: This randomized prospective study was initiated to clarify whether individualized heparin and protamine dosing has immediate effects on hemostatic activation and platelet function in adult cardiac surgery. METHODS: Sixty adults undergoing elective coronary artery bypass grafting (CABG) were assigned to receive individualized heparin and protamine (HMS group, n= 29) or a standard dose (ACT group, n=24). Measures of thrombin generation and Multiplate (Verum Diagnostica, Munich, Germany) platelet function tests were performed before and after cardiopulmonary bypass (CPB). RESULTS: HMS patients received higher heparin (p = 0.006) and lower protamine (p<0.001) doses. Post-CPB, HMS managed patients showed significantly lower thrombin generation (thrombin-antithrombin (TAT) p<0.02) than the ACT group. Moreover, HMS managed patients had a better preservation of platelet function (COL p = 0.013; ADP p = 0.04; TRAP p = 0.04). CONCLUSION: An individualized and stable heparin concentration and appropriate dosing of protamine can reduce thrombin generation and preserve platelet function, even in short-time CPB.

Surgical site infections after coronary artery bypass graft surgery: incidence, perioperative hospital stay, readmissions, and revision surgeries.

PURPOSE: High-tech operations performed in cardiac surgery are associated with an increased risk of surgical site infections. In this study, we investigated if surgical site infections following cardiac surgery influence revision surgeries and patients' length of stay, and compared the results to German hospital infection surveillance data. METHODS: Over a period of 3 years, 2,621 patients of a cardiac surgery unit were enrolled following cardiac artery bypass graft surgery. Patients were examined for the incidence of surgical site infections, revision surgeries, and length of stay. The results were compared to the National Reference Center (NRC) data retrospectively. RESULTS: Of the observed population, 4.5 % suffer from surgical site infections, and in 7.7 % of the patients, revision surgery had to be performed. The length of stay was exceeded significantly for the patients with surgical site infections (average stay 14.5 vs. 42.2 days, p < 0.001). Compared to the NRC data, severe surgical site infections were not increased significantly. CONCLUSION: Surgical site infections resulted in revision surgeries with a significantly increased inpatient stay. However, this increase did not differ significantly from comparable German university hospitals.

RAGE influences obesity in mice. Effects of the presence of RAGE on weight gain, AGE accumulation, and insulin levels in mice on a high fat diet.

BACKGROUND: The metabolic syndrome is defined by the presence of obesity, insulin resistance, dyslipidemia, and hypertension. Advanced glycation end products (AGEs) are stable end products of the Maillard reaction, whereby AGE accumulation is considered not only a biomarker of aging but is also associated with several degenerative diseases. AGEs are recognized by several receptor molecules of which the receptor of AGEs (RAGE) is currently the most intensively studied receptor. Activation of RAGE causes an unfavorable proinflammatory state and deletion of RAGE in diabetic animals has been reported to protect against atherosclerosis. AGEs and a high fat diet are associated with cardiovascular diseases, whereas is still not clear whether a direct link between high fat nutrition and AGEs exists in vivo. MATERIALS AND METHODS: C57BL/6 and C57BL/6 RAGE -/- mice were fed a high fat diet to induce obesity. Weight, insulin, lipid levels, AGE modifications, and cardiac gene expression were analyzed. RESULTS: The absence of RAGE resulted in accelerated weight gain, increased plasma cholesterol, and higher insulin levels in obese mice. The hearts of normal and obese RAGE -/- mice contained lower levels of the AGE arginine-pyrimidine and 3DG-imidazolone than RAGE + / + animals. RAGE -/- mice also exhibited lower expression of the genes encoding the antioxidative enzymes MnSOD, Cu/ZnSOD, and ceruloplasmin in cardiac tissue, whereas the AGE receptors AGER-1, -2, and -3 were equally expressed in both genotypes. Obese mice of both strains expressed increased amounts of AGER-2. Only obese RAGE + / + mice exhibited a reduced mRNA accumulation of Cu/Zn SOD. CONCLUSION: These data suggest that RAGE is involved in the development of obesity and insulin resistance.

Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a 70,000 Mr protein that has been isolated from the membrane of red cells. The function of DAF is to inhibit the assembly of amplifying enzymes of the complement cascade on the cell surface, thereby protecting them from damage by autologous complement. We raised monoclonal antibodies to DAF and used them to study its distribution in cells from the peripheral blood of normal individuals and of patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the unusual susceptibility of red cells to the hemolytic activity of complement. The results of immunoradiometric assays and of fluorescence-activated cell sorter analysis showed that DAF was present not only on red cells but was widely distributed on the surface membrane of platelets, neutrophils, monocytes, and B and T lymphocytes. By Western blotting, we observed small but consistent differences in the Mr of DAF from the membranes of various cell types. Quantitative studies showed that phagocytes and B lymphocytes, which presumably enter more frequently in contact with immune complexes and other potential activators of complement, had the highest DAF levels. As previously reported by others, the red cells from PNH patients were DAF deficient. When the patients' red cells were incubated in acidified serum (Ham test), only the DAF-deficient cells were lysed. In addition, we detected defects in DAF expression on platelets and all types of leukocytes. The observed patterns of DAF deficiency in these patients were consistent with the concept that the PNH cells were of monoclonal origin. In one patient, abnormal and normal cells were found only in the erythroid, myeloid, and megakaryocytic lineages. In two other patients, the lymphocytes were also DAF deficient, suggesting that a mutation occurred in a totipotent stem cell. It appears, therefore, that the lesion leading to PNH can occur at various stages in the differentiation of hematopoietic cells.

Deficiency of lymphocyte function-associated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. Functional correlates and evidence for a phosphatidylinositol membrane anchor.

Lymphocyte function-associated antigen 3 (LFA-3) is a widely distributed cell surface glycoprotein that binds to the T lymphocyte CD2 surface glycoprotein. This interaction mediates CTL-target cell conjugate formation and adhesion of thymocytes to thymic epithelial cells. CD2 is also the E rosette receptor of T lymphocytes and mediates rosetting with autologous E by binding to LFA-3. We describe deficient expression of LFA-3 on E from paroxysmal nocturnal hemoglobinuria (PNH) patients. PNH is an acquired defect affecting phosphatidylinositol-anchored membrane proteins, of which decay-accelerating factor (DAF) is most important in the clinical symptoms of PNH. LFA-3-negative, weakly positive, and positive populations were found among PNH E. There was a good correlation with DAF deficiency. PNH E exhibited decreased binding of 125I-CD2 and rosetting with a human T lymphoma cell line. PNH E readily incorporated purified LFA-3, restoring LFA-3 expression and the CD2 binding and rosetting activity to normal levels. The expression of DAF was not restored after the incorporation of purified LFA-3 into PNH E, showing that LFA-3 and DAF are different molecules. Phosphatidylinositol-specific phospholipase C (PIPLC) treatment of a B lymphoma cell line released 35% of the cell surface LFA-3 and 62% of DAF. LFA-3 on E was resistant to PIPLC. However, when LFA-3 purified from human E was reconstituted in sheep E or human E and subjected to PIPLC treatment, 40-50% of LFA-3 was released from the cell membrane. The results show that LFA-3 is attached to the cell membrane by a phosphatidylinositol glycolipid moiety, and confirm previous findings (37-41) that LFA-3 is a cell adhesion molecule that mediates adhesion by interacting with CD2 antigen.

5-methyltetrahydrofolic acid is not a methyl donor for biogenic amines: enzymatic formation of formaldehyde.

Contrary to previous reports, 5-methyltetrahydrofolic acid does not mediate the methylation of dopamine to epinine. Instead, this methyl donor is degraded enzymatically to formaldehyde, which condenses with dopamine to form a tetahydoisoquinoline derivative. The latter has chromotographic characteristics very similar to those of epinine, which likely accounts for the original misidentification of the product.

Phytohemagglutinin: inhibition of the agglutinating activity by N-acetyl-D-galactosamine.

The effect of simple sugars on the agglutinating activity of phytohemagglutinin was studied. N-Acetyl-d-galactosamine selectively inhibits the agglutination of leukocytes and erythro- cytes by phytohemagglutinin.

DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts.

Drug development is needed to improve chemotherapy of patients with locally advanced or metastatic colon carcinoma, who otherwise have an unfavorable prognosis. DNA topoisomerase I, a nuclear enzyme important for solving topological problems arising during DNA replication and for other cellular functions, has been identified as a principal target of a plant alkaloid 20(S)-camptothecin. Significantly increased concentrations of this enzyme, compared to that in normal colonic mucosa, were found in advanced stages of human colon adenocarcinoma and in xenografts of colon cancer carried by immunodeficient mice. Several synthetic analogs of camptothecin, selected by tests with the purified enzyme and tissue-culture screens, were evaluated in the xenograft model. Unlike other anticancer drugs tested, 20(RS)-9-amino-camptothecin (9-AC) induced disease-free remissions. The overall drug toxicity was low and allowed for repeated courses of treatment.

Heterogeneity of 5'-nucleotidase activity in lymphocytes in chronic lymphocytic leukemia.

The specific activity of 5'-nucleotidase was determined in lymphocyte plasma membranes from 14 normal subjects and 10 patients with chronic lymphocytic leukemia (CLL). Whereas the enzyme was present in the preparation from normal lymphocytes, in 7 out of 10 CLL patients the membranes had markedly decreased or no detectable 5'-nucleotidase activity. The lack of this activity from the lymphocytes of most patients with CLL constitutes an alteration in a plasma membrane enzyme from the normal cell. The presence of the enzyme in the lymphocytes of some patients with CLL and its decrease in others provide further evidence for biochemical heterogeneity among patients with this disorder.

Tocopherol transport in the rat erythrocyte.

The transport of vitamin E (alpha-tocopherol) has been studied in the rat erythrocyte in vivo and in vitro. Uptake and efflux are independent of energy, but sensitive to temperature. Tocopherol is localized to the cell membrane. Rapid exchange takes place between erythrocytes and serum with an hourly fractional tocopherol efflux of 26%. The vitamin is transferred from the erythrocyte to the low density lipoproteins. These experiments indicate that tocopherol, like cholesterol, is a constituent of the erythrocyte membrane which is in dynamic equilibrium with the corresponding plasma compound.

Human lymphocytes: 5'-nucleotidase-positive and -negative subpopulations.

The enzyme, 5'-nucleotidase (5'N) (E.C.-3.1.3.5) is present in lymphocytes isolated from the blood of normal subjects. This activity is markedly decreased or not detectable in the cells from three-quarters of patients with chronic lymphocytic leukemia (CLL), while supranormal levels are found in less than 10% of the cases. To determine whether the decreased 5'N value in CLL represents a lower activity per cell or fewer enzyme-containing cells than in the normal, conditions were established for the histochemical measurement of 5'N in human lymphocytes. It was found that the cells isolated from the blood of normal subjects or patients with CLL consist of 5'N-positive and 5'N-negative subpopulations. Normal subjects who had high 5'N specific activity were shown to have a greater percentage of 5'N-positive cells than individuals with low 5'N activity. Patients with CLL who had no activity by standard chemical assay had no 5'N-positive cells, while the exceptional patient with CLL with a higher than normal specific activity showed an percentage of 5'N-positive cells. It is suggested that the selective proliferation of 5'N-positive and 5'N-negative populations may account for the heterogeneity of 5'N in CLL.

Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse.

The level of nucleoside deaminase was determined in extracts of mouse tissues obtained during a period of accelerated erythropoiesis induced by hypoxia, hemorrhage, or the injection of phenylhydrazine. Under these conditions a striking (10- to 100-fold) elevation of the enzyme activity occurred in the spleen. Similar results were obtained with the injection of purified erythropoietin. In control animals, only a trace of nucleoside deaminase activity was detected in the blood. During the reticulocyte response which followed erythropoietic stimulation, there was a sharp increase in the blood level of nucleoside deaminase, which rose up to 120 times that of control animals. By differential centrifugation, the enzyme was localized to the reticulocyte-rich fraction. Erythrocyte nucleoside deaminase remained elevated even after the reticulocyte count had fallen to normal in the phenylhydrazine-treated mice or to zero after the cessation of hypoxia. There was a very gradual decline in the enzyme activity in the blood which fell to the barely detectable control levels about 45 days after the initial reticulocyte response, a time period which corresponds to the survival of the mouse red blood cell. The persistence of high levels of nucleoside deaminase for the full life span of a generation of erythrocytes formed during stress, viewed in contrast to the virtual absence of the enzyme from normal erythrocytes of all ages, represents an enzymatic difference between the normal red blood cell and the cell produced under conditions of accelerated erythropoiesis.

Adenosine deaminase activity in chronic lymphocytic leukemia. Relationship to B- and T-cell subpopulations.

The level, phenotypes, and isozyme distribution of adenosine deaminase (ADA) were determined in lymphocytes from patients with chronic lymphocytic leukemia (CLL). The ADA level in lymphocytes from patients with untreated CLL was consistently lower than in lymphocytes from normal subjects. No significant differences were found in the phenotype or isozyme distribution. In untreated patients, the ADA level was inversely correlated with the lymphocyte count and the percentage of bursa-equivalent (B) cells. After therapy, a diminution in the lymphocyte count was associated with an increase of ADA activity towards normal levels. The ADA levels were slightly higher in the thymus-derived (T) than in the B lymphocytes from normal subjects. The B cells had lower activity than T cells in patients with CLL. They also had a lower activity than the B cells from normal subjects. The ADA level was 2.3-fold higher in T cells from patients with CLL than in normal T cells. The decrease in ADA levels is an anomaly that is reversible and appears to be a reflection of the proliferation of abnormal B cells in this disorder.

Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes exhibit abnormalities in decay accelerating factor (DAF), acetylcholinesterase, and resistance to autologous C5b-9 attack. To investigate the nature of the lesion underlying PNH cells, we examined the relationship of these abnormalities to one another. Analyses of DAF in acetylcholinesterase-negative erythrocytes revealed that these two abnormalities involve functionally independent molecules, coincide precisely in the same cell populations, and are similarly expressed in PNH II and more complement-sensitive PNH III erythrocytes. The DAF and acetylcholinesterase deficiencies contrast with the C3b/C4b receptor (CR1) deficit, which is less profound and similarly distributed in complement-insensitive cell populations. Hemolytic studies showed that defective resistance to autologous C5b-9 attack is mediated by another mechanism. Whereas reconstitution of PNH II erythrocytes with DAF completely corrected their complement sensitivity, DAF reconstitution of PNH III erythrocytes restored their ability to circumvent C3b uptake but had no effect on their heightened susceptibility to reactive lysis. Assays of complement-insensitive (PNH I) erythrocytes surviving after reactive lysis disclosed partial DAF and acetylcholinesterase deficits. These findings indicate that the PNH lesion involves multiple membrane components and that PNH I erythrocytes are also abnormal.

Advanced glycation endproducts: a biomarker for age as an outcome predictor after cardiac surgery?

OBJECTIVE: A decline in the function of all organs can be detected during ageing. Although the trend appears to be stable, deviation within the elderly population is much greater in comparison to young controls. The aim of the study was to identify a marker of senescence which correlates to heart function. Advanced glycation endproducts (AGEs) accumulate with age and are associated with degenerative diseases. METHODS: Carboxymethyllysine (CML) concentrations in the pericardial fluid (as a measure of AGEs) were analysed with ELISA technique in 75 patients undergoing cardiac surgery and correlated with clinical parameters and outcome of these patients. RESULTS: CML content of pericardial fluid increases significantly with age. AGEs show an inverse correlation to left ventricular ejection fraction. High CML levels correlate with poor outcome of patients as shown by adverse cardiac events, prolonged ventilation time and prolonged stay within the Intensive Care Unit. Within all parameters, AGE concentration of the pericardial fluid fits better with the outcome of the patients in comparison to age alone. Interestingly, medical treatment with nitrates correlates with increased CML content. CONCLUSION: AGEs, in addition to being a marker of senescence, appear to represent a prognostic factor in cardiac surgery, which can be used as a predictor of patient outcome.

Peste Des Petits Ruminants in Benin: Persistence of a Single Virus Genotype in the Country for Over 42 Years.

Peste des petits ruminants (PPR) is a contagious and often fatal disease affecting sheep and goats. Currently, it is endemic in Africa, the Middle and Near East, the Indian subcontinent and China. Understanding the molecular epidemiology and evolution of PPR virus (PPRV) can assist in the control of the transboundary spread of this economically important disease. We isolated PPRV from pathological and swab samples collected 42 years apart (1969 and 2011) in Benin, West Africa, and sequenced the full genome of two isolates (Benin/B1/1969 and Benin/10/2011). Phylogenetic analysis showed that all of the characterized isolates clustered within viral lineage II and that the 2011 isolates fell into two distinct subgroups. Comparison of the full genome sequences revealed a 95.3% identity at the nucleotide level, while at the protein level, the matrix protein was the most conserved between the two viruses with an identity of 99.7% and only one amino acid substitution over the 42-year sampling period. An analysis of specific amino acid residues of known or putative function did not identify any significant changes between the two viruses. A molecular clock analysis of complete PPRV genomes revealed that the lineage II viruses sampled here arose in the early 1960s and that these viruses have likely persisted in Benin since this time.

Adriamycin damage: in vivo repair by lymphoma cells.

The in vivo repair of ADR-induced cell damage was investigated in the DBA3 transplantable mouse lymphoma. After a single injection of 5 or 15 mg ADR/kg body weight into DBA/2J mice, the survival fraction of clonogens showed a 2.2- to 4.4-fold decrease at 12 or 18 hours post injection and returned to pretreatment levels within 6 hours. These changes were accompanied by the appearance and disappearance of DNA crosslinks and breaks. Because cell division and/or cell loss could not explain the return of clonogens to pretreatment level, the results strongly suggest repair of ADR damage in tumor cells in situ. Such an efficient repair mechanism, responding to a high toxic dose of ADR, constitutes a therapeutically unfavorable event that may contribute to drug resistance.